Our goal is to understand how multi-gene clusters are organized and regulated. Specifically, 1) how does the physical organization influence expression of the genes, 2) what are the mechanisms that operate to 'open' chromatin, and 3) how is a particular gene within an open cluster chosen for expression? The model evolving from studies of the human beta-globin cluster is that control elements upstream of the genes are essential for locus activation (chromatin opening). These upstream elements also increase the expression of the nearest available genes, with availability chiefly determined by the promoter. This model is supported by the genotype phenotype correlations observed in patients with deletional beta-thalassemia and hereditary persistence of fetal hemoglobin. To examine the contribution of the upstream hypersensitive sites and beta-A 3' enhancer in the regulation of each of the globin genes in the intact cluster, we introduced the entire chicken beta-globin cluster into mice. All four transgenes were expressed with the correct developmental pattern, suggesting that the stage-specific erythroid transcriptional milieus existed before the divergence of aves and mammals. Deletion of either the upstream hypersensitive sites or of the beta-A 3' enhancer caused reduced expression of all four globin transgenes. Thus, all of the genes are under the control of both the upstream hypersensitive sites and the enhancer and the influence of the control elements can extend beyond the nearest active gene. Each of the four hypersensitive sites found upstream of the cluster was tested for enhancer activity. HS2 and HS3 showed significant enhancer activity and HS2 was studied in detail. Four binding sites for the GATA-1 transcription factor were found and accounted for the enhancer activity. We have also sequenced the cluster. This revealed four gene conversions and allowed deduction of the order of gene duplication. CR1 repetitive elements accounted for 16% of the cluster, a surprisingly large fraction, suggesting that the cluster is a hot spot for CR1 insertion.